The goal of this research program is to identify the mechanisms by which axons drive Schwann cell myelination. We had previously reported that type III NRG1 is the long sought instructive signal that determines the ensheathment fate of axons and does so, in part, via activation of PI3 kinase in Schwann cells. To elucidate how type III NRG1, and PI3 kinase, drive Schwann cell myelination, we had proposed to: i) examine the specificity and cleavage of NRG1 isoforms, ii) characterize which of the downstream effectors of PI3 kinase promote Schwann cell ensheathment and myelination, and iii) determine whether NRG1 signals are required to maintain the integrity of the axon myelinating Schwann cell unit in the adult. We have made important progress on each of these aims, in particular by providing evidence that Akt, a key effector of PI3 kinase, has an essential role in promoting Schwann cell myelination. We have also obtained evidence that Akt activity is concentrated in the paranodes and have identified a series of candidate Akt substrates that are markedly upregulated with myelination. In this revision, we will build on these findings by: i) determining if NRG1 is the key activator of Akt in the paranodes, ii) further characterize the functional consequence of inhibiting Akt activity, and iii) identify Akt substrates in myelinating Schwann cells as a prelude to their functional analysis. These studies should provide major insights into how the axon drives formation of the myelin sheath and may have important implications for elucidating the pathogenesis of peripheral neuropathies. PUBLIC HEALTH RELEVANCE: The long term goal of this research program is to identify the signals on axons, and the downstream pathways they activate in Schwann cells, that determine whether nerve fibers are ensheathed or myelinated. Recent data from our lab indicates that the type III isoform of the neuronal growth factor, neuregulin-1 (NRG1), provides the long sought instructive signal that drives myelination of axons. Binding of type III NRG1 to Schwann cell (erbB) receptors activates PI3 kinase, a key signaling molecule essential for ensheathment and myelination. A major PI3 kinase effector, Akt, plays a critical role in myelination. To extend these studies, and our understanding of how Akt promotes myelin assembly, we propose to: i) determine whether NRG1 signaling is required for the compartmentalized activation of Akt by analyzing nerve fibers from mice in which NRG1 has been conditionally ablated, ii) characterize further the effects of inhibiting Akt signaling on myelination by expressing dominant negative Akt constructs in Schwann cells in vitro, and iii) identify Akt substrates in Schwann cells by SILAC and mass spectrometry. These studies build upon key findings of the role of Akt signaling during myelination made with support of the parent grant. They will provide important new insights into the mechanisms by which NRG1 signaling promotes myelination and, potentially, help elucidate the pathogenesis of dysmyelinating peripheral neuropathies.